Tobacco products

ABSTRACT

A tobacco product includes a dry powder composition comprising an amount of myo-inositol effective for treating and/or delaying onset of dysplastic lesions in the bronchial airway of an individual. A method for administering myo-inositol to an individual comprises the individual consuming the tobacco product.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/894,087 filed Feb. 12, 2018, which is a divisional application ofU.S. application Ser. No. 13/642,878 filed Apr. 8, 2013, now U.S. Pat.No. 9,895,321, which is a 371 of PCT/US2011/033587 filed Apr. 22, 2011,and claims priority to U.S. Applications Nos. 61/327,323 filed Apr. 23,2010 and 61/439,799 filed Feb. 4, 2011.

FIELD OF THE INVENTION

The present invention relates to methods and products for treatingand/or delaying onset of dysplastic lesions.

BACKGROUND OF THE INVENTION

There are certain highly contagious diseases that, despite theavailability of vaccines, continue to be serious health problems indeveloping and, in some cases, developed countries. For example, measlesis a highly contagious human disease caused by the measles virus (MV),and vaccination programs have dramatically reduced its incidence.However, despite the success of global measles vaccination programs,measles was still responsible for an estimated 345,000 deaths in 2005,with most of these deaths occurring in developing countries. Measlesoutbreaks also continue to occur in developed countries that have failedto maintain a high level of population immunity. Global vaccinationcoverage is approximately 80% but, according to WHO estimates, more than23 million infants did not receive their first dose ofmeasles-containing vaccine in 2007. Tuberculosis is also a highlycontagious disease that, despite the availability of vaccines, continuesto be a serious health problem. Many significant developments have beenmade in the field of human papillomavirus (HPV) vaccines. See, forexample, the Garcea et al US 6,165,471, 7,763,259, 2009/0229535 and2011/0033893. Additionally, HPV vaccines are now commercially available.However, their use in developing countries is hampered by conditionsnecessary for administration. Safety, disposal, and wastage issuesassociated with using current lyophilized vaccines that requirereconstitution with clean water and use of needles for injection remaina concern and limit widespread coverage in certain countries. Newvaccine formulations for diseases such as these that are more easilyadministered, stable at ambient temperatures, easily transported, andcost effective would be beneficial.

Aerosol delivery of measles vaccines have been employed and overcome atleast some of the disadvantages of needle-administered vaccines. Severalclinical studies have been performed using aerosol measles vaccinationby nebulizing commercial lyophilized formulations after reconstitution,and this route of administration resulted in equal or better immuneresponses in children greater than 10 months of age compared withinjection. Additionally, dry powder vaccine formulations adapted foradministration by inhalation are disclosed in the Sievers et al U.S.Patent Application No. 2008/0035143 A1 and employ carriers such amyo-inositol and/or maltodextrin. Such dry powder formulations areadvantageous in avoiding needle delivery and the need for reconstitutingwater, and may be supplied in unit dosage form to avoid waste. However,additional means for facilitating effective vaccination are desired aswell.

SUMMARY OF THE INVENTION

The present invention is directed to, inter alia, vaccines, methods ofadministering vaccines, optionally with adjuvants, methods for providingstorage stable unit dosages of vaccine, methods and products fortreating and/or delaying onset of dysplastic lesions, wafers for oraladministration.

More specifically, in one embodiment, the present invention is directedto methods of treating and/or delaying onset of dysplastic lesions inthe bronchial airway of an individual. The methods comprise deliveringto the respiratory track of the individual an inhalable dry powdercomposition including an effective amount of myo-inositol.

In another embodiment, the invention is directed to a tobacco productwhich includes a dry powder composition comprising an amount ofmyo-inositol effective for treating and/or delaying onset of dysplasticlesions in the bronchial airway of an individual tobacco smoker.

In another embodiment, the present invention is directed to additionalmethods of treating and/or delaying onset of dysplastic lesions in thebronchial airway of an individual. These methods comprise sublinguallydelivering a wafer comprising an effective amount of myo-inositol to theindividual.

In yet another embodiment, the present invention is directed to methodsfor administering a vaccine to an individual. The methods compriseadministering a first portion of the vaccine to the individualsublingually and administering a second portion of the vaccine to theindividual by an administration route other than sublingually, whereinthe first portion and the second portion combined provide an effectiveamount of the vaccine.

In a further embodiment, the present invention is directed to additionalmethods for administering a vaccine to an individual. The methodscomprise administering a first portion of the vaccine to the individualvia a first route comprising sublingually, orally to thegastrointestinal tract, inhalation, or injection, and administering asecond portion of the vaccine to the individual by an administrationroute other than the first route, wherein the first portion and thesecond portion combined provide an effective amount of the vaccine.

In another embodiment, the present invention is directed to a vaccine inthe fonn of an inhalable dry powder composition comprising a combinationof nanoparticles having a diameter less than 1 μm and microparticleshaving a diameter greater than 2.0 μm, wherein the dry powdercomposition comprises at least about 10% particles having a diameter notgreater than about 3 μm, at least about 10% particles having a diameterof about 5-10 μm, and at least about 10% particles having a diameter ofat least about 20 μm.

In another embodiment, the invention is directed to a wafer comprising acompressed dry powder composition comprising myo-inositol, wherein thewafer has a thickness of not greater than about 2 mm and dissolves inwater at room temperature in less than one minute. The wafer is suitablefor oral administration of therapeutic agents.

In another embodiment, the invention is directed to a wafer comprising acompressed dry powder composition comprising a vaccine and a carrier,wherein the wafer has a thickness of not greater than about 2 mm anddissolves in water at room temperature in less than about one minute.

In another embodiment, the invention is directed to methods of providingstorage stable unit dosages of a vaccine. The methods compriseindividually packaging unit dosages of a dry powder compositioncomprising the vaccine and a carrier in blister compartments formed ofgas and moisture resistant material.

The various embodiments of the invention provide improved methods andproducts for administration of therapeutic agents, including, but notlimited to vaccines, and may provide advantages of conventional methodsand/or products.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description will be more fully understood in viewof the Drawings, in which:

FIG. 1 sets forth a schematic view of a Carbon dioxide-AssistedNebulization with a Bubble Dryer (CAN-BD) process which may be used toform dry powder compositions for use in the present invention;

FIG. 2 is a scanning electron micrograph (SEM) showing a CAN-BD drypowder composition of 90% myo-inositol/10% leucine;

FIG. 3 is a SEM showing the surface of a highly compressed wafer diskformed from the dry powder composition by compression at 2000 psi for 15minutes;

FIG. 4 shows the stability of measles vaccine virus potency for ameasles vaccine dry powder formulation in blister packaging and capsulepackaging over a 34 month period, as described in Example 4.

FIGS. 5A and 5B show powder mass and measles vaccine virus titerdistribution on the short-stack Andersen Cascade Impactor (ACI) Stages0, 1, 3 and 5, as described in Example 4.

FIG. 6 is a scanning electron micrograph (SEM) showing a CAN-BD drypowder composition as described in Example 6, prepared according to thefirst protocol.

FIG. 7 is a scanning electron micrograph (SEM) showing a CAN-BD drypowder composition as described in Example 6, prepared according to thesecond protocol.

DETAILED DESCRIPTION

The methods and products of the present invention, in certainembodiments, employ dry powder compositions. In one embodiment, the drypowder compositions may be formed by the techniques disclosed in theSievers et al U.S. Pat. No. 6,630,121, incorporated herein in itsentirety by reference, which techniques are employed in the commerciallyavailable Carbon dioxide-Assisted Nebulization with a Bubble Dryer(CAN-BD), although the dry powder compositions may be formed by othertechniques known in the art, as well. CAN-BD is a closed, continuousprocess wherein dense carbon dioxide (for example, supercritical or nearsupercritical carbon dioxide) and a liquid aqueous solution orsuspension are intimately mixed in a low volume mixing tee at roomtemperature and very high pressure (for example about 80 bar), and theresulting emulsion mixture is rapidly expanded through a flow restrictor(for example, having an inside diameter of about 75 to 380 μm) into adrying chamber at or near atmospheric pressure to generate aerosols ofmicrobubbles and microdroplets. Warm nitrogen gas is used to maintainthe drying chamber at near ambient temperatures, i.e., below about 60°C., to dry the aerosols and generate dry powders containing less than 5weight percent water. In many cases, the dry powders contain less than2%, less than 1% or less than 0.5% by weight water. Typically, the drypowder compositions will include a carrier, for example, myo-inositol,sucrose, lactose, mannitol, sorbitol, trehalose, one or more aminoacids, peptides, polylactic-glycolic acid (PLGA), polylactic acid (PLA),lecithin, dispalmitoylphosphatidylcholine (DPPC), maltodextrin, or thelike.

In a specific embodiment of the CAN-BD for use in preparing dry powdercompositions for various embodiments of the present invention, awater/oil/water (w/o/w) emulsion is mixed with dense carbon dioxide.Water-in-oil-in-water (w/o/w) emulsions are a multiple emulsion systemin which the internal and external aqueous phases are separated by anoily lipophilic layer. The w/o/w system consists of internal aqueousdrops surrounded by a surfactant film, with an aqueous layer with asurfactant boundary film in turn surrounding these internal drops.Typically, for drug preparations, the drug is dissolved in the inneraqueous phase, poly (lactic-co-glycolic acid) (PLGA) is dissolved in avolatile organic solvent, for example, dichloromethane, ethyl acetate oracetone, forming the oily phase and an emulsifier is dissolved in theouter aqueous phase. Use of the CAN-BD system causes rapid eliminationof the volatile organic phase and polymer (PLGA) precipitation and soliddrug-loaded microspheres are formed upon drying. In a more specificembodiment, the w/o/w emulsion comprises a drug dissolved or suspendedin water/polylactic-glycolic acid in dichloromethane or ethyl acetate asan organic phase/water with stabilizing polyvinyl alcohol.

In certain embodiments of the invention, the dry powder compositions maycomprise myo-inositol (cis-1,2,3,5-trans-4,6-cyclohexanehexol). Drypowder compositions comprising myo-inositol are disclosed in the Sieverset al U.S. Patent Application No. 2008/0035143 A1, incorporated hereinin its entirety, although other myo-inositol dry powder compositions,including those generally and/or specifically disclosed herein are alsosuitable for use in embodiments of the present invention. Dry powdercompositions comprising myo-inositol may optionally further include oneor more of amino acids, for example, arginine, alanine, histidine,leucine, and/or methionine, surfactants, for example, palmitic acid,stearic acid, Tween 20, Tween 80, and/or Pluronic F68, stabilizers,buffers, for example, tricine, sodium or potassium phosphate, sodiumacetate, and/or sodium citrate, metal chelating agents, for example EDTAand/or DTPA, gelatin, albumin, or the like. In one specific embodiment,myo-inositol and leucine are employed in a 98.5:1.5 weight ratio.Advantageously, myo-inositol dry powder compositions containing one ormore additional components are provided using the CAN-BD process. It hasbeen discovered that when pure myo-inositol is processed by the CAN-BDprocess at a temperature of about 50° C., a crystal polymorph which isdifferent from the common crystal polymorph of native myo-inositolforms. This polymorph has an exothermic transition with onset of110-130° C. and appears to be thermally stable at room temperature foronly short periods of time (less than 2 weeks), but remains for longerperiods of time when stored at temperatures of 2-8° C. (for at least 3months and up to 5 months), and is removed by annealing at 115° C. orabove. The moisture content of this new polymorph is very low (0.1%water) and identical to the moisture content of native myo-inositol. TheX-ray diffraction pattern of this polymorph differs from those of nativemyo-inositol and a previously disclosed myo-inositol dihydrate (Bonnet2006). Processing pure myo-inositol using the CAN-BD process attemperatures at 60° C. and above provides the common crystal polymorphof native myo-inositol.

In specific embodiments, the dry powder compositions of myo-inositolcomprise a fine particle fraction (as measured according to the AndersenCascade Impactor (ACI) and USP Chapter <601>) of less than about 5.8 μmof at least about 20%, and in a more specific embodiment, a fineparticle fraction less than about 3.3 μm of at least about 10%. Infurther embodiments, the dry powder compositions of myo-inositolcomprise a fine particle fraction of less than about 5.8 μm ranging fromabout 35 to about 50%, and a fine particle fraction less than about 3.3μm ranging from about 15 to about 30%. In a specific embodiment, thesedry powder compositions of myo-inositol further include leucine, forexample, in a myo-inositol:leucine weight ratio in a range of from about90.0:10.0 to about 99.5:0.5.

In a first embodiment, the invention is directed to methods of treatingand/or delaying onset of dysplastic lesions in the bronchial airway ofan individual. In a specific embodiment, the individual is a tobaccosmoker. Within the present disclosure and claims, the term “tobaccosmoker” is used to refer to current and former tobacco smokers.Cigarette smoke is a dominant cause of lung cancer, but the damagecaused by cigarette smoke is not limited solely to the lung. Rather,cigarette smoke forms a field of injury throughout the entirerespiratory tract. Mild to moderate dysplasia in the form of dysplasticlesions in the respiratory tract represent an early abnormal change inthe cytology of cells that is non-cancerous but is often considered aprecancerous state. Lam et al, Cancer Epidemiol. Biomarkers Prev.,15:1526-1531 (2006) previously studied the use of myo-inositol for lungcancer chemoprevention wherein smokers and former smokers with mild tomoderate airway dysplasia were treated with myo-inositol for threemonths by oral administration of doses up to 30 grams per day to thegastrointestinal tract, with the maximum tolerated dose being 18 gramsper day. A significant regression in dysplastic lesions was observed.Gustafson et al, Science Translational Medicine, 2(26) 26ra25:1-11(2010) have determined that airway P13K pathway activation is an earlyand reversible event in lung cancer development and concluded thatmyo-inositol is a P13K inhibitor and its lung cancer chemopreventionproperties may be related to this inhibition.

The present methods therefore employ myo-inositol in treating and/ordelaying onset of dysplastic lesions in the bronchial airway of anindividual. In one embodiment, the methods comprising delivering to therespiratory track of the individual an inhalable dry powder compositionincluding an effective amount of myo-inositol. The inhalable powder may,but is not required to, be prepared according to the CAN-BD techniquesdescribed above. The effective amount will be dependent on the extent ofany existing dysplasia and the condition of the individual to betreated. One of ordinary skill in the art ill appreciate that thetargeted therapy of the present methods will advantageously allow theuse of effective doses which are generally lower than the systemicadministration employed by Lam et al. In one embodiment, the effectiveamount comprises from about 0.01 to about 10 grams per day, and in morespecific embodiments, can be from about 0.5 to about 10, from about 1 toabout 10, from about 0.01 to about 5, from about 0.5 to about 5, fromabout 1 to about 5, grams per day. The dry powder composition mayinclude one or more excipients, for example, surfactant, amino acids, orthe like, to improve the particle formation method, particle size, orthe like.

The dry powder composition may be delivered to the respiratory tract byinhalation through the mouth or through the nose, or both. In a specificembodiment, in order to provide a dry powder composition which will beeffective for delivery throughout the respiratory tract for treatment orpreventive treatment, the dry powder composition comprises a fineparticle fraction as measured according to the Andersen Cascade Impactor(ACT) of less than about 5.8 μm of at least about 20%, and in a morespecific embodiment, a fine particle fraction less than about 3.3 μm ofat least about 10%. In a further embodiment, the dry powder compositioncomprises a fine particle fraction of less than about 5.8 μm rangingfrom 35 to 50%, and a fine particle fraction less than about 3.3 μmranging from 15 to 30%.

The dry powder composition may optionally further include one or moreadditional active agents. In one specific embodiment, the additionalactive agent comprises an adjuvant, an analgesic, anti-cancer agent,antimicrobial agent, vaccine, antiviral agent, antifungal agent,antibiotic, immune suppressant, thrombolytic, anticoagulant, centralnervous system stimulant, decongestant, diuretic vasodilator,antipsychotic, sedative, anesthetic, anti-inflammatory, antioxidant, orantihistamine, or a combination of two or more thereof. In a morespecific embodiment, the additional active agent comprises ananti-cancer agent. In a yet more specific embodiment, the anti-canceragent may comprise a prostanoid such as iloprost, beraprost, ortreprostinil, as disclosed in the Keith et al U.S. Patent ApplicationNo. 2006/027654.6 A1, which is incorporated herein in its entirety.

In additional embodiments, an additional amount of myo-inositol isdelivered to the individual in a form other than an inhalable drypowder, either prior to or following the delivery of the inhalablepowder composition comprising myo-inositol example, the additionalamount of myo-inositol may be delivered orally to the gastrointestinaltract of the individual, or by sublingual administration. In a specificembodiment, the additional amount of myo-inositol is delivered bysublingual administration of a wafer comprising myo-inositol. In aspecific embodiment, suitable waters comprising Myo-inositol areprepared by compression of a dry powder composition comprisingmyo-inositol as described in further detail below.

In one embodiment, the dry powder composition comprising an effectiveamount of myo-inositol is provided in a tobacco product, for example ina cigarette or cigar, or in a smokeless tobacco chew product. In aspecific embodiment, the dry powder composition is provided in a tobaccoproduct, for example, a cigarette, in a location adapted for inhalationof the composition during use of the product. For example, in acigarette, the dry powder composition is provided in a filter, a porousmesh compartment, for example, a porous mesh bag, or other area whichallows inhalation of the composition during smoking of the cigarette todeliver the composition to the individual's respiratory track.

In another embodiment of the inventive methods of treating and/ordelaying onset of dysplastic lesions in the bronchial airway of anindividual, the methods comprise sublingually delivering a wafercomprising an effective amount of myo-inositol as detailed above to theindividual. Within the present specification and claims, the term“wafer” refers to a relatively thin unit dose form suitable forsublingual application. In specific embodiments, the water has athickness less than about 2 mm, or, more specifically, a thickness ofabout 1 mm. The wafer may be in the shape of a disk, square, ellipsoid,banana, or other configuration, as desired, and may have a diameter orlength and width dimensions in the range of about 4-10 mm. In oneembodiment, the wafer has a disk configuration with a diameter of about6-8 mm and a thickness of about 1 mm.

In a specific embodiment, the wafer comprises a compressed dry powder ofmyo-inositol as described above, which, in one embodiment, may be formedaccording to the CAN-BD process. The wafer may include one or moreexcipients in addition to the dry powder composition, as desired.Excipients traditionally used by the pharmaceutical industry andGenerally Regarded As Safe (GRAS) in the formation of oral tablets maybe used. The excipients are typically blended with the myo-inositolbased dry powder and compressed. For example, one or more binding agentsand/or disintegration agents may be employed. Suitable binding agent ordisintegration agents include, but are not limited to, pelleting gradesucrose, sorbitol, lactose, trehalose, mannitol, starch, guar gum,methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, polyethylene glycol, maltodextrin, andthe like. Conventional additives such as mold release agents, forexample magnesium stearate, muco adhesive agents, for example chitosan,flavoring agents, and the like, may also be employed. Typically, suchwafers may be formed by pressing with conventional equipment at suitablepressures. For example, suitable pressures, are in the range of greaterthan 1 psi, or more specifically, greater than about 10 psi. In specificembodiments, the wafers may be formed by pressing with conventionalequipment at pressures in a range of about 10 to less than about 100psi, more specifically about 10 to about 80 psi, or about 10 to about 50psi, or at pressures greater than about 100 psi, or up to about 500,about 1000 or about 2000 psi, i.e., in the range of about 500-2000 psi,or, more specifically, about 1000 psi. In cases where one or moresensitive biological materials are included in the wafer, for example,an enveloped virus vaccine, lower pressures in the range of about 1 psito about 500 psi, more specifically, about 10 to about 100 psi, arepreferable.

The wafers which are administered sublingually will desirablydisintegrate in saliva to disperse the myo-inositol. In one embodiment,the wafers dissolve in wateroo temperature in less than about oneminute. In another embodiment, the wafers dissolve in saliva in fromabout 15 to 30 seconds.

Additional wafers within the scope of the invention will be describedhereafter as well.

In the methods of treating and/or delaying the onset of dysplasticlesions in the bronchial airway of an individual by sublinguallydelivering a wafer comprising an effective amount of myo-inositol, it isalso within the scope of the present invention to include an additionalstep of delivering an additional amount of myo-inositol to theindividual by an administration route other than sublingually. Forexample, in one embodiment, the additional amount of myo-inositol isdelivered orally to the gastrointestinal tract of the individual.

The myo-inositol wafer employed in the methods of treating and/ordelaying onset of dysplastic lesions may, if desired, further include anadditional active agent. In a specific embodiment, the additional activeagent comprises an adjuvant, an analgesic, anti-cancer agent,antimicrobial agent, vaccine, antiviral agent, antifungal agent,antibiotic, immune suppressant, thrombolytic, anticoagulant, centralnervous system stimulant, decongestant, diuretic vasodilator,antipsychotic, sedative, anesthetic, anti-inflammatory, antioxidant, orantihistamine, or a combination of two or more thereof. In a morespecific embodiment, the additional active agent comprises ananti-cancer agent, in a yet more specific embodiment, the anti-canceragent may comprise a prostanoid such as iloprost, beraprost, ortreprostinil, as disclosed in the Keith et al U.S. Patent ApplicationNo. 2006/0276546 A1.

In another embodiment, the present invention is directed to methods foradministering a vaccine to an individual. In specific embodiments, thevaccines described in the present specification may be vaccines for avirus or bacteria. The methods employ combination administration whichoffer benefits that a single administration route or form may not. Forexample, the combination administration methods of the invention offeradditional transport, deposition, and/or dissolution resulting ininduction of an immune response in several modes. Therefore, an immuneresponse can occur through more than one system in the body, i.e., inthe respiratory tract and a gastrointestinal or injection initiatedplasma system. Delivery to various locations in the body induce combinedresponses, i.e., both humoral and mucosal immunity. Any suitable vaccinemay be administered in the methods of the invention. In a specificembodiment, the vaccine comprises a vaccine for a virus or bacteria, andin a more specific embodiment, the vaccine comprises a measles vaccine,a rubella vaccine, a tuberculosis vaccine, a human papillomavirusvaccine, an influenza vaccine, a mumps vaccine, a yellow fever vaccine,a rotavirus vaccine, a cholera vaccine, a Pseudomonas aeruginosavaccine, an anthrax vaccine, or a combination of two or more thereof. Ina more specific embodiment, the vaccine comprises a measles vaccine. Inanother specific embodiment, the vaccine comprises a measles vaccine anda rubella vaccine in combination. In yet another embodiment, the vaccinecomprises one or more tuberculosis (TB) vaccines. Further, the vaccinemay include an adjuvant if desired. Vaccine adjuvants are known in theart and may include, but are not limited to, ovalbumin, MF59microemulsion, polylactic-glycolic acid (PLGA), CpG oligonucleotides,saponin and saponin derivatives such as QS21, alum, Freund's completeadjuvant, B30-MDP, LA-15-PH, monophosphoryl/Lipid A (MPL), and the like.Alum has been widely used as an adjuvant in injected vaccines but hasnot been approved for inhalation.

In a first embodiment, the methods for administering a vaccine to anindividual using a combination administration comprise administering afirst portion of the vaccine to the individual sublingually andadministering a second portion of the vaccine to the individual by anadministration route other than sublingually, wherein the first portionand the second portion combined provide an effective amount of thevaccine. The combined administration provides good immunity and mayallow reduced dosages and/or improved immunity effects. The second routeof administration can be oral administration to the gastrointestinaltract, inhalation, i.e., through the mouth or nose or both, orinjection, for example intravenous, subcutaneous, intramuscular, or thelike. In one embodiment, the second portion of the vaccine is in theform of an inhalable dry powder composition and is administered to theindividual by inhalation through the mouth. In another embodiment, thesecond portion of the vaccine is in the form of an inhalable dry powdercomposition and is administered to the individual by inhalation throughthe nose.

In a second embodiment, the methods for administering a vaccine to anindividual using a combination administration comprise administering afirst portion of the vaccine to the individual via a first routecomprising sublingually, orally to the gastrointestinal tract,inhalation, or injection, and administering a second portion of thevaccine to the individual by an administration route other than thefirst route, wherein the first portion and the second portion combinedprovide an effective amount of the vaccine. In a specific embodiment,the first route comprises sublingually and the second route comprisesinhalation. In more specific embodiments, the first route comprisessublingually and the second route comprises inhalation, and the vaccinecomprises measles vaccine or human papillomavirus vaccine. In anotherspecific embodiment, the first route comprises inhalation and the secondroute comprises injection, and more specifically, the first routecomprises inhalation and the second route comprises injection, and thevaccine comprises human papillomavirus vaccine. In further specificembodiments, the first route comprises sublingually and the second routecomprises orally to the gastrointestinal tract. In any of theembodiments wherein one of the routes of administration is sublingually,the vaccine may be in the form of a wafer. The wafer may optionally bein the form of any of the wafers described herein, although other wafersmay be employed as well. Water administration may provide the benefit ofnot requiring cold chain handling for distribution and administration.Additionally, administration of a wafer does not require skilled medicalpersonnel. In any of the embodiments wherein one of the routes ofadministration is by inhalation, any of the dry powder compositions asdescribed herein may be employed, although other dry powder compositionsmay be employed as well.

In a third embodiment, the methods for administering a vaccine to anindividual using a combination administration comprise administering avaccine in the form of an inhalable powder including both nanoparticlesand microparticles that are sized for delivery to various locations inthe body to induce combined responses, i.e., both humoral and mucosalimmunity. More specifically, in one specific embodiment, the vaccine inthe form of an inhalable dry powder composition comprises a combinationof nanoparticles having a diameter less than 1 μm and microparticleshaving a diameter greater than 20 μm. In a more specific embodiment, thedry powder composition comprises at least about 10% particles having adiameter not greater than about 3 μm, at least about 10% particleshaving a diameter of about 5-10 μm, and at least about 10% particleshaving a diameter of at least about 20 μm, and, in a more specificembodiment, further comprises at least about 10% particles having adiameter of about 3-5 μm. Any of the aforementioned dry powdercompositions may be employed, although other dry powder compositions maybe employed as well. Further, any of the aforementioned vaccines may beemployed, although other vaccines may be employed as well. In specificembodiments, the vaccine comprises a measles vaccine, alone or incombination with a rubella vaccine. In a further embodiment, the drypowder composition comprises myo-inositol.

In another embodiment, the present invention is directed to waferscomprising a compressed dry powder composition comprising myo-inositol.The myo-inositol may be employed as an active ingredient, for examplefor use in a method of treating and/or delaying onset of dysplasticlesions in the bronchial airway, wherein the myo-inositol is used in aneffective amount for the indicated therapy. Alternatively, themyo-inositol may be used as a carrier for an active agent, for example,for use in therapeutic methods wherein myo-inositol is not an effectiveactive agent. In a specific embodiment,the wafer has a thickness of notgreater than about 2 mm and dissolves in water at room temperature inless than about one minute.

Whether or not the myo-inositol is employed as an active agent, thewafers as described may also include one or more additional ingredientswhich are active agents. Exemplary active agents include, but are notlimited to, an adjuvant, an analgesic, anti-cancer agent, antimicrobialagent, vaccine, antiviral agent, antifungal agent, antibiotic, immunesuppressant, thrombolytic, anticoagulant, central nervous systemstimulant, decongestant, diuretic vasodilator, antipsychotic, sedative,anesthetic, anti-inflammatory, antioxidant, or antihistamine, or acombination of two or more thereof. In a specific embodiment, the activeagent comprises a vaccine. In a more specific embodiment, the vaccinecomprises a vaccine for a virus or bacteria, and in a more specificembodiment, the vaccine comprises a measles vaccine, a rubella vaccine,a tuberculosis vaccine, a human papillomavirus vaccine, an influenzavaccine, a mumps vaccine, a yellow fever vaccine, a rotavirus vaccine, acholera vaccine, a Pseudomonas aeruginosa vaccine, an anthrax vaccine,or a combination of two or more thereof. In a more specific embodiment,the active agent comprises a measles vaccine, alone or in combinationwith a rubella vaccine.

In another embodiment, the present invention is directed to a wafercomprising a compressed dry powder composition comprising a vaccine anda carrier, wherein the wafer has a thickness of not greater than about 2mm and dissolves in water at room temperature in less than about 10minutes, or, more specifically, less than about 5 minutes, less thanabout 2 minutes, or less than about one minute. In a specificembodiment, the vaccine comprises a vaccine for a virus or bacteria, andin a more specific embodiment, the vaccine comprises a measles vaccine,a rubella vaccine, a tuberculosis vaccine, a human papillomavirusvaccine, an influenza vaccine, a mumps vaccine, a yellow fever vaccine,a rotavirus vaccine, a cholera vaccine, a Pseudomonas aeruginosavaccine, an anthrax vaccine, or a combination of two or more thereof. Ina more specific embodiment, the vaccine comprises a measles vaccine. Thewafer may comprise, for example, myo-inositol sucrose, mannitol, ortrehalose. In a specific embodiment, the dry powder composition isprepared according to the CAN-BD process, and has the fine particlefractions and low moisture content as described above.

The vaccine-containing wafers promote rapid dissolution of the vaccineand restore propagation and virus or virus-like particle proteinimplantation in the gingiva, buccal, and tongue mucosal cells, as wellas the mandibular lymph nodes. Any one or a combination of thesedelivery sites can be expected to induce an immune response to provideprotection against the disease, for example, measles, rubella, HPV,especially the 4 main serotypes of human papillomavirus, and/or multipleinfluenza virus strains.

The aforementioned wafers may include one or more excipients in additionto the dry powder composition, as desired. In order to formulate avaccine that takes the shape of a sublingual wafer, for example, whencompressed using a pellet press, the dry powder which is originallydesigned to be free-flowing is advantageously combined with selectedexcipients that demonstrate adhesive behavior, but do not negativelyimpact the effectiveness or potency of the vaccine. Excipientstraditionally used by the pharmaceutical industry and Generally RegardedAs Safe (GRAS) in the formation of oral tablets may be used. Theexcipients are typically blended with the myo-inositol based dry powderand compressed. For example, one or more binding agents and/ordisintegration agents may be employed. Suitable binding agent ordisintegration agents include, but are not limited to, pelleting gradesucrose, starch, guar gum, methyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose (HPMC), hydroxyethyl starch (HES), cornstarch (high amylase), polyvinyl pyrrolidone (PVP), polyethylene glycol(PEG), polyvinyl alcohol, microcrystalline cellulose (MCC),carboxymethylcellulose (CMC), sorbitol, and the like, and combinationsthereof. In a specific embodiment, a combination of HPMC, PVP and MCC isemployed in combination with myo-inositol. This combination hasadvantageously been found to provide sufficient binding to allow waferformation at lower pressures, i.e., less than about 100 psi. This is animportant feature as many vaccine viruses may be inactivated ifcompressed at higher pressures typically employed in wafer formation.Moreover, this combination provides wafers that advantageouslydisintegrate in water or saliva in less than about 1 minute. In a morespecific embodiment, a wafer comprises 20% HPMC, 10% PVP, 20% MCC and50% myo-inositol, based on the combined weight of these fouringredients. In another specific embodiment, sorbitol is employed.Conventional additives such as mold release agents, for examplemagnesium stearate, muco adhesive agents, for example chitosan,flavoring agents, for example menthol, and the like, may also beemployed. For example, small percentages of one or more muco-adhesivematerials such as chitosan to foster adhesion of the wafers on desiredmucosal sublingual sites may be employed. Typically, the wafers may beformed by pressing with conventional equipment at suitable pressures,i.e., in the range of greater than 10 psi. In specific embodiments, thewafers may be formed by pressing with conventional equipment atpressures in a range of 10 to less than 100 psi, more specifically 10 to80 psi, or 10 to 50 psi, or at pressures greater than 100 psi, or up to500, 1000 or 2000 psi. The wafers may be opaque or transparent,depending on the particle size distribution, the composition of theoriginal powder, and the amount of powders to which various pressuresare applied.

In another embodiment, the present invention is directed to methods ofproviding storage stable unit dosages of a vaccine. Within the presentspecification and claims, the term “storage stable” refers to theability of the vaccine to continue to exhibit potency as determined by astandard plaque assay or the 50% cell culture infective dose (CCID₅₀)assay wherein virus is added to cells cultured in liquid media inmicrotiter plate wells. Both the plaque and CCID₅₀ assays are describedin Kisich et al, Vaccine, 29:905-912 (2011), incorporated herein byreference. A single test unit consists of the entire cell layer in awell. After the virus is added to test units, it is serially diluteddown the microtiter plate, and each test unit is scored as infected ornot infected and the virus titer is determined by the Spearman-Karbermethod for calculation of CCID₅₀ (see Ballew, In Clinical VirologyManual; Specter, S.; Lancz, G. J., Eds.; Elsevier: New York, 1986, p187, and Finney, Charles Griffin & Company Ltd: London, 1964, p 524).The methods according to the invention comprise individually packagingunit dosages of a dry powder composition comprising the vaccine andmyo-inositol in blister compartments formed of gas and moistureresistant material. The dry powder compositions contain less than 5weight percent water. In many cases, the dry powder compositions containless than 2%, less than 1%, or less than 0.5% by weight water. Beyondpreserving powder aerosolization and deposition properties,stabilization via residual moisture reduction is an important factor inpreserving potency by preventing microorganism growth and otherdegradation. The blister compartments fonned of gas and moistureresistant material insure that the moisture content remains low and thatthe vaccine is not exposed to ultraviolet radiation, oxidants or othercontaminants.

In specific embodiments of the methods, the unit dosages are stable forat least 1 year when stored at 2-8° C., or, more specifically, arestable for at least 2 years when stored at 2-8° C. In one specificembodiment, the unit dosages comprise a measles vaccine which is stablefor at least 1 year when stored at 2-8° C., or, more specifically, arestable for at least 2 years when stored at 2-8° C. In additionalembodiments, the gas and moisture resistant material comprises alaminate including at least one aluminum foil layer and at least onepolymer film layer, for example, of a polymer having oxygen, carbondioxide and/or moisture transmission resistance. The vaccine may be anysuitable vaccine, including, but not limited to a vaccine for a virus orbacteria, and in a more specific embodiment, the vaccine comprises ameasles vaccine, a rubella vaccine, a tuberculosis vaccine, a humanpapillomavirus vaccine, an influenza vaccine, or a Pseudomonasaeruginosa vaccine, an anthrax vaccine, or a combination of two or morethereof. In one specific embodiment, the dry powder composition is in aninhalable form, while, in another embodiment, the dry powder compositionis in a compressed wafer form.

In a further embodiment, the present invention is directed to dry powdercompositions comprising an antibiotic for tuberculosis, morespecifically, capreomycin, kanamycin and/or isoniazid. These dry powdercompositions may be employed in combined administration methods asdescribed above. In one embodiment, the powders are formed by the CAN-BDprocess and, optionally, may be employed in methods of providing storagestable unit dosages by individually packaging unit dosages of the drypowder composition in blister compartments formed of gas and moistureresistant material. The dry powder compositions contain less than 5weight percent water. In many cases, the dry powder compositions containless than 2%, less than 1%, or less than 0.5% by weight water. In aspecific embodiment, the dry powder compositions comprise myo-inositolas a carrier. The particle size requirements and doses for the TB drugdry powder composition requires microparticles targeted largely to thealveolar space, where Mtb enters in the early stages of infection andalso to poorly vascularized lesions and granulomas which harbor bacilliin protective microenvironments that often elude conventional therapy.Powders according to the invention having tine particle fractions asgreat as about 20% less than 3.3 μm aerodynamic diameter are ideal foralveolar deposition. By depositing antibiotics in the deep lung, one maybe able to reach TB lesions that lack a strong blood supply, and mayalso increase the targeted dose to the lung airspace and tissue whilereducing systemic side effects and, potentially, the length oftreatment. Using inhalable antibiotics in combination with moretraditional treatments of oral administration, injection and the like,provides a two-pronged attack strategy for MDR-TB. Increasing thetargeted dose in the lung airspace and targeting bacteria in protectivemicroenvironments such as those in granulomas may prevent thedevelopment of MDR-TB by ensuring that threshold doses of antibioticsreach bacteria populations that are difficult to target by traditionalmethods.

Various aspects of the present invention are illustrated in thefollowing Examples.

EXAMPLE 1

The myo-inositol employed in the various methods and products describedherein has been studied to evaluate its safety and immunogenicity in aninhalable dry-powder measles vaccine when administered in a single doseto Rhesus monkeys. The dry powder inhaler, PuffHaler®, as disclosed bySievers et al, was utilized, and the animals were dosed once byinhalation delivery. The control group received a marketed measlesvaccine via subcutaneous injection. Animals in the treatment groupreceived 50 mg of dry powder measles vaccine via PuffHaler® inhalation.All vaccinated animals developed protective levels of neutralizingantibodies (>120 mIU/ml). Animals remained healthy throughout the courseof the study; no effects on clinical observations, body weights,respiration rates, or breathing patterns were noted. The monkeys weresubsequently sent to Johns Hopkins University where they were challengedwith live measles virus; neither clinical symptoms nor viremia wereobserved in animals immunized by inhalation through face masks.

EXAMPLE 2

In this example, inhalable dry powder compositions comprisingmyo-inositol were produced using the CAN-BD process as described aboveand as set forth in FIG. 1. The process conditions for the CAN-BD wereas follows:

-   CO₂ pressure: 1500 psi-   Sample concentration: 10 wt %-   Sample Flow rate: 0.3 ml/min-   N2 outlet drying temperature: 70° C.-   N2 flow rate: 35 l/min-   Secondary drying: 30 mins @70° C.

In order to improve the powder attributes for use as inhalable powder,⁻various amino acids dispersing agents were employed. Leucine, arginine,and alanine were initially screened at 5 wt % for their effects inimproving the myo-inositol powder dispersibility. Powders were analyzedfor particle properties (fine particle fractions, size and moisturecontent) and the leucine-containing formulation was selected as the bestperforming powder. Additional formulations were prepared to furtheroptimize the myo-inositol-leucine powder, by varying the leucine amount(see Table 1). The rationale was to produce an inhalable myo-inositolpowder with minimum amounts of dispersing agent. Powders werecharacterized for their fine particle fraction (Andersen CascadeImpactor, ACI), moisture content (Karl Fischer titration) and sizedistribution, and results are presented in Table 1. Based on theresults, myo-inositol containing 1.5 wt % leucine was selected as afinal formulation. This formulation was successfully processed andrepeated under smaller batch (2 g) and larger batch (10 g) processingconditions to determine reproducibility. All the powders were preparedunder the same processing conditions described above, similar to thoseused for inhalable live measles vaccine dry powders.

TABLE 1 Batch ACI Batch size, Myo- Powder <5.8 <3.3 Moisture No. ginositol Leucine Yield μm μm Content 1 2 95   5 wt %   50% 47% 17% 2 1.699   1 wt % 71.5% 55% 25% 0.15% ± 0.01 3 1.5 97.5 2.5 wt %   70% 0.23% ±0.01 4 1.5 99.5 0.5 wt % 10%  5% 0.11% ± 0.03 5 10.8 99   1 wt %   74%51% 20%  0.2% ± 0.01 6 15 98.5 1.5 wt % 57.5% 52% 15% 0.2%

Myo-inositol: leucine (weight ratio 98.5: 1.5) dry powder was alsotested for its moisture uptake and powder performance under highhumidity conditions. Briefly, 10 mg powder replicates were placed in anopen blister and exposed to 70% relative humidity (RH) for 15 mins. Nomoisture uptake was observed and the sample had exactly the samemoisture content before and after exposure. The moisture content ofpowder before and after exposure was measured as 0.22 wt %. Also, nochange was observed in the fine particle fractions of the powders asmeasured Andersen Cascade Impactor, before and after 70% RH exposure.

EXAMPLE 3

In this example, thin wafers according to the present invention wereformed. A dry powder composition prepared earlier by CAN-BD from 98.5%myo-inositol and 1.5% leucine generally in accordance with the techniquedescribed in Example 2 was used. Wafers having a thickness of about 1min. in the form of rigid cylindrical disks, 7 millimeters in diameter,were prepared from the dry powder composition, or from Dominopelleting-grade sucrose, using a hand-held KBr pellet press fromInternational Crystals Laboratories of Garfield, N.J., using a pressureof about 1000 psi for 1 minute. Solid rigid brittle wafers were formed.Additional wafers were prepared using a 1&2 Ton E-Z Pellet Press or aPort-A-Press. With the pressure gauge integrated into this equip en aspossible to monitor applied pressure continually throughout theexperiment. The wafers dissolved in less than one minute in water t roomtemperature. The wafers made from the 98.5% myo-inositol-1.5% leucinedry powder composition were transparent, as were wafers made from ameasles virus vaccine placebo dry powder composition formed using 50grams/L of myo-inositol in the CAN-BD process.

FIG. 2 is a scanning electron micrograph (SEM) showing of a CAN-BD drypowder composition of 90% myo-inositol/10% leucine, while FIG. 3 is aSEM showing the surface of a highly compressed wafer disk formed fromthe dry powder composition by compression at 2000 psi for 15 minutes.Flat glassy areas and some particles are shown at the surface.

EXAMPLE 4

In this example, a measles-containing dry powder composition wasprepared using the CAN-BD process as described and a measles formulationcomprising Edmonston-Zagreb measles vaccine virus, myo-inositol,gelatin, arginine-HCl, histidine, alanine, lactalbumin hydrosylate,tricine buffer and additional ingredients from the Minimum EssentialMedium (MEM) vaccine virus harvest fluid. Before drying, the aqueousformulations contained 11% total dissolved solids in water, and thesolids comprised 50 g/L myo-inositol, 25 g/L hydrolyzed gelatin, 16 g/Larginine-HCl, 1.0 g/L alanine, 2.1 g/L histidine, 3.5 g/L lactalbuminhydrolysate, and 3.0 g/L tricine, with a pH of 6.5-7.0. The dry powdercomposition had a moisture content less that 3%.

Unit dosages of the dry powder composition were stored in moisture andgas resistant blister packaging formed of aluminum foil/polymer laminateand in moisture and gas resistant capsules. The blisters and capsuleswere stored in foil pouches together with molecular sieve desiccantpacks to maintain a very low humidity environment. The unit dosages werestored as described at 2-8° C. for almost 3 years and demonstratedexcellent stability according to the CCID₅₀ assay. The results are setforth in FIG. 4 which shows the measles vaccine dry powder in blisterslost about 0.8 log CCID₅₀ of potency after 34 months and the measlesvaccine dry powder in capsules lost about 0.7 log CCID₅₀ of potencyafter 34 months. These results demonstrate that the measles vaccine drypowder can be quite stable for a commercially acceptable refrigeratedshelf life.

An additional experiment was conducted using samples of theblister-packaged dry powder compositions and a PuffHaler® dry powderinhaler device from Aktiv-Dry LLC. For each experiment, 3blister-packaged unit dosages were sequentially dispersed into ashort-stack ACI configuration (Stages 0, 1, 3, 5, and F). The mass ofpowder that impacted on each stage was first determined gravimetricallyand then the powder fractions on Stage 0 (>9.0 μm in aerodynamicdiameter), Stage 1 (5.8 μm to 9.0 μm), Stage 3 (3.3 μm to 5.8 μm), andStage 5 (1.1 μm to 3.3 μm) were recovered in a sterile manner usingphosphate buffered saline (PBS). The mass of powder on the final glassfiber filter Stage F (<1.1 μm) could be weighed, but could not bereliably recovered using PBS. The vaccine virus titer in each of thesesamples was measured by CCID₅₀. As shown by the mass distribution inFIG. 5A, most of the measles vaccine dry powder particles collected onthe ACI stages were in the respirable size range, i.e., <5.8 μm. Of theaverage 31 mg total of measles vaccine dry powder in the threePuffHaler® blisters used in each experiment, 28% 4% (s.d.) was recoveredin the mass fraction <5.8 μm, and 12%+3% in the fraction <3.3 μm. Inthis case, a single 10 mg measles vaccine dose contained an average of3.3 log CCID₅₀, of vaccine virus titer in the respirable fraction (<5.8μm). Furthermore, the vaccine titer was uniformly distributed in thefine and course microparticles. The mass normalized titer did not changewithin experimental error as a function of the particle size bins asshown in FIG. 5B. These findings are very important, as they confirm thepotency per unit mass of particles sized throughout the respirablerange.

EXAMPLE 5

A HPV-containing dry powder composition was also prepared using theCAN-BD process as described and the myo-inositol/leucine (98.5/1.5 wtratio) and HPV16L1 capsid-based vaccine. The dry powder compositionexhibited a fine particle mass fraction of 50%<5.8 μm (ACI). Unitdosages of the dry powder composition were stored in moisture and gasresistant blister packaging formed of aluminum foil/polymer laminate.SDS-PAGE analysis of the composition after processing and after the unitdosages were stored at 2-8° C. for 1 year showed no detectabledegradation of HPV capsid protein.

EXAMPLE 6

This example demonstrates the preparation of solid microspheres usingthe CAN-BD system and a water/oil/water emulsions system.

A first protocol, adapted from Lamprecht et al, International Journal ofPharmaceutics, 184(0:97-105 (1999), employed an inner water phaseconsisting of 500 mg of isoniazid in 10 mL of phosphate buffered saline(PBS) or distilled water. The 500 mg of isoniazid is 0.24% by mass ofthe entire w/o/w emulsion (210 mL including all three phases) Theorganic phase consists of 500 mg of PLGA (50:50) dissolved in 40 mL ofethyl acetate (EA). The first, internal water-in-oil (w/o) emulsion iscreated by homogenizing the isoniazid water phase and the EA organic or“oily” phase at 5000 RPM for 3 minutes. The external water phasecontains 2% polyvinyl alcohol (PVA) w/v, with respect to the externalwater phase of 200 mL. To create the w/o/w emulsion, the 10 mL of thepreviously described w/o emulsion is poured into the external aqueousPVA phase and is homogenized on ice at 8000 RPM for 3 min. Afterhomogenization the w/o/w volume expands from 210 mL to 450 mL with twophases—a foam phase and a liquid phase. The foam phase has beenevaluated by SEM and does not contain any significant concentration ofparticles. The liquid phase is poured into the product solution cylinderof the CAN-BD system where the emulsion was dried via at 60° C., 30L/min N₇, 1260 psi, with a flow rate of 0.5 mL/min. This procedure wasused to prepare microspheres from emulsions containing about 0.2-0.8isoniazid, about 0.2-0.8 and about 1-2% PVA. The resulting particles arespherical with dimples and range in size from about 0.25 μm to about 7μm. Microspheres produced using an emulsion containing 0.19% isoniazid,0.24% PLGA and 1.92% PVA with PBS, EA and water are shown in FIG. 6.

In a second protocol, adapted from Song et al, Colloids and Surfaces A:Physicochem. Eng. Aspects, 276:162-167 (2006), 100 mg of PLGA (75:25) isdissolved in 10 mL of EA. The organic phase is added to 20 mL of anaqueous phase containing the stabilizer PVA. The PVA aqueous phase is 1%w/v with respect to the 20 mL water phase. The mixture is allowed tocome to saturation and is then emulsified for one minute with a probetip sonicator operating at setting six and between 1-10% intensity.While sonicating, the external aqueous phase which consists of 80 mL ofwater and dissolved drug is added to the w/o emulsion created by thesonication of the PLGA/EA phase and the water PVA phase. Aftersonication 30 mL of the w/o/w emulsion is processed via the CAN-BDsystem at 0.5 mL/min, 30 L/min N₂, 60° C. and between 1200 and 1260 psi.The particles made using protocol 3 employed bovine serum albumin (BSA)as a model protein are shown in FIG. 7. These particles are generallyrange in size from about 0.25 μm to about 7 μm.

The various examples and embodiments described herein are illustrativein nature only and are non-limiting of the invention defined by theclaims. Additional embodiments of the invention within the scope of theclaims will be apparent to one of ordinary skill in the art in view ofthe present disclosure.

What is claimed is:
 1. A tobacco product, including a dry powdercomposition comprising an amount of myo-inositol effective for treatingand/or delaying onset of dysplastic lesions in the bronchial airway ofan individual.
 2. The tobacco product of claim 1, in the form of acigarette.
 3. The tobacco product of claim 2, wherein the dry powdercomposition is provided in a location adapted for inhalation uponsmoking of the cigarette.
 4. The tobacco product of claim 2, wherein thedry powder composition is provided in a filter in the cigarette.
 5. Thetobacco product of claim 2, wherein the dry powder composition isprovided in a porous mesh compartment.
 6. The tobacco product of claim1, in the form of a cigar.
 7. The tobacco product of claim 1, in theform of a smokeless tobacco chew product.
 8. The tobacco product ofclaim 1, wherein the dry powder composition further includes anadditional active agent comprising an adjuvant, analgesic, anti-canceragent, antimicrobial agent, vaccine, antiviral agent, antifungal agent,antibiotic, immune suppressant, thrombolytic, anticoagulant, centralnervous system stimulant, decongestant, diuretic vasodilator,antipsychotic, sedative, anesthetic, anti-inflammatory, antioxidant, orantihistamine, or a combination of two or more thereof.
 9. The tobaccoproduct of claim 4, wherein the additional active agent comprises ananti-cancer agent.
 10. A method for administering myo-inositol to anindividual, comprising the individual consuming the tobacco product ofclaim
 1. 11. The method of claim 10, wherein from about 0.01 to about 10grams of myo-inositol is administered per day.
 12. The method of claim10, wherein from about 0.5 to about 10 grams of myo-inositol isadministered per day.
 13. The method of claim 10, wherein from about 1to about 10 grams of myo-inositols administered per day.
 14. The methodof claim 10, wherein about 0.01 to about 5 grams per day of myo-inositolis administered per day.
 15. The method of claim 10, wherein from about0.5 to about 5 grams per day of myo-inositol is administered per day.16. The method of claim 10, wherein from about 1 to about 5 grams perday of myo-inositol is administered per day.